Cholera is a disease of pandemic proportions, and its health, economic, and political impacts cannot be overstated. V. cholerae is primarily associated with aquatic habitats; however, under appropriate conditions, V. cholerae can infect the human host and grow to very high densities within the human intestine. The transition to the human host represents a major environmental shift for V. cholerae. V. cholerae must sense and respond to this new environment in order to survive, colonize, and produce disease. Regulation of colonization and virulence factors in response to environmental cues is mediated through several regulatory factors, including the highly conserved RNA- binding global regulatory protein CsrA. CsrA plays a role in quorum sensing, biofilm formation, virulence gene expression, carbon metabolism, and environmental fitness, both in V. cholerae and in other bacterial species. We recently demonstrated that V. cholerae CsrA regulates the master virulence gene regulator ToxR in response to particular amino acids in the medium, consistent with a role for CsrA in incorporating environmental cues into the complex regulation of virulence gene expression. CsrA is essential for the growth of V. cholerae, making it difficult to study in the laboratory; however, we have isolated a csrA point mutant strain that is viable, but defective for ToxR regulation. Using this mutant, we showed that csrA is critical for virulence in the infant mouse model of V. cholerae infection. Despite its importance as regulator of virulence and other processes, there is little or no information about what genes are directly targeted by CsrA in V. cholerae. The goal of this proposal is to define the direct targets of CsrA regulation. Because csrA is essential, and because CsrA is a posttranscriptional regulator, traditional genetic approaches are limited. We have developed a novel technique involving in vivo CsrA-RNA co-immunoprecipitation followed by deep sequencing of the immunoprecipitated RNA species to identify the direct targets of CsrA regulation. This information, combined with RNA-Seq transcriptome analysis, will be critical for understanding the larger network of genes controlled by CsrA in this pathogen. We also propose to solve the crystal structure of V. cholerae CsrA bound to target RNA. There are currently no published structural analyses of CsrA among the vibrionaceae. Understanding the structural features of V. cholerae CsrA will greatly increase our knowledge of how CsrA post-transcriptionally regulates its RNA targets.